MicroRNAs (miRNAs) are a recently identified class of small regulatory RNA molecules found in most organisms, including human. miRNAs are encoded by the genomes and processed into ~ 22-nucleotide products which are thought to play important regulatory role, mainly through degrading target messenger RNAs (mRNAs) and/or inhibiting translation of protein-coding mRNAs. Interestingly, many miRNAs are expressed in a tissue-specific manner, suggesting their potential roles in regulating tissue-specific gene expression. However, given more than 400 miRNAs identified in mammals, the molecular mechanisms and the in vivo functions of most miRNAs remain unknown. In our preliminary studies, we found that miR-1, miR- 133, miR-206 and miR-208 are muscle-specifically expressed (and therefore referred to as "muscle miRNAs"). Ectopic expression or knockdown of miR-1 and miR-133 in myoblasts modulates their proliferation and differentiation processes, indicating that miRNAs are importance for animal development and function. The long-term goal of our research is to understand the molecular mechanisms that control development and function of cardiac muscle. The discovery of miRNAs opened a completely new field to investigate how miRNAs may participate in "classical" gene expression pathways. Our central hypothesis is that miRNAs are components of the molecular circuitry that controls mammalian cardiac development and function. The overall goal of this proposal is to explore the molecular mechanisms of miRNA function, using cardiac muscle as our model system. In particular, we will study the in vitro and in vivo function of cardiac- specific miR-208 and identify its regulatory targets which are involved in cardiac muscle differentiation and development. In addition, we will assess the global role of miRNAs in heart development, using Dicer conditional knockout mice. The specific aims are: Aim #1. To study the in vitro and in vivo role of miR- 208 in heart development and cardiac gene expression. Aim #2. To define the global role of miRNA- mediated regulation in cardiac muscle using Dicer conditional knock-out mice. Aim #3. To identify and experimentally test the regulatory targets of miRNAs in the heart. Identification of the regulatory mRNA targets of miRNAs in the heart will be the key to the understanding of the molecular mechanisms of miRNA function. We will use a combination of computational prediction and experimental confirmation to study the regulatory targets repressed by miRNAs in the heart. Our studies will provide important insights into the molecular mechanisms behind miRNAs that control mammalian heart development and cardiac gene expression. The molecular strategies revealed in these studies may apply to pathophysiologically- related cardiac muscle events such as human congenital heart defects (CHD), cardiac failure and cardiac hypertrophy.